Preparation of virus vaccines



United States Patent 3,060,004 PREPARATIGN @11 VRUS VAMIINES Ray M.Butcher, Belchertown, Ralston B. Read, In, Sunderiand, and WarrenLitsiry, Amherst, Mass. (all University of Massachusetts, Amherst,Mass.) No Drawing. Filed Apr. 7, 1959, Ser. No. 804,569 4 Ciaims. (Cl.167-78) This invention relates to a process for the preparation of virusvaccines and to the improved virus vaccines so produced. The inventionbroadly involves the inactivation of living virus microorganisms at hightemperatures for very short periods of time.

A virus vaccine is usually defined as a suspension of inactivated orattenuated microorganisms. A suspension of living virus microorganismshas the ability to produce an immunity in man or animal but its use isaccompanied by the danger of producing the disease. The use ofattenuated or partially inactivated virus vaccines is also not withoutdanger because of the possibility that the microorganisms containedtherein may also produce diseases. The killed or inactivated type ofvirus vaccine is generally harmless when properly administered, and itsvalue depends On its ability to develop immunity.

The present invention provides a process for the preparation ofinactivated virus vaccines which can be used to provide suitableimmunity against virus diseases. The invention also includes a processfor the preparation of partially inactivated virus vaccines which can beused to obtain lowered virulence with a subsequent reduction indeleterious response of the animal to the vaccine. The process of thisinvention can also be used to prepare inactivated viral antigens for usein serological diagnostic testing as well as for preparing inactivatedviral antigens to be used in the subsequent preparation of specificantiserum in man and animals to be used either in serological diagnosticprocedures or in therapeutic passive imrnuniza tion procedures in bothman and animals.

The invention broadly comprises the heating of live virus suspensions athigh temperatures for very short periods of time. The hightemperature-short time heating provides an advantageous method ofinactivating live virus suspensions thus inactivating specificpathogenic agents with a minimum of destructive eflfect upon itsantigenic structure.

Difierent types of equipment can be used to perform the rapid heattreatment of the virus suspensions such as the heat exchanger describedin the article entitled Come-Up Time Method of Milk Pasteurization, byRead, Boyd, Litsky, and Hankinson, Journal of Milk and Food Technology,volume 19, No. 2, February 1956. The heat exchanger described in thispublication consists of a stainless steel pressure tank or reservoir anda small bore stainless steel tube. The tube is electrically heated andthe virus suspension to be treated is forced through the small bore tubeby air pressure at a rate so as to insure turbulent flow as determinedby the Reynolds number and is heated by heat exchange from the smallbore tube.

EXAMPLE 1 Newcastle disease Virus (9251 strain) was used in thisexample. The virus had been passaged 125 times in emice bryonatedchicken eggs at the time of receipt and was passaged 3 more times priorto testing.

The pools of allantoic fluid containing virus were prepared from tenoreleven-day-old embryonated eggs inoculated with a 1:10 or 1:100 dilutionof infective allantoic fluid. The pools of virus were frozen in afreezer prior to use. Both hemagglutination and EID titrations wereperformed on each pool after thawing and prior to testing.

The embryonated eggs used in this example were PPLO and pullorum free.All chickens used for vaccination purposes were hatched in thelaboratory from eggs collected from known Newcastle disease-free birds.All birds were tested serologically prior to vaccination.

All chicken cells for hemagglutination titrations were obtained frombirds maintained in the laboratory for that specific purpose and theblood was washed 3 times with 0.85 percent sodium chloride prior to use.Fresh cells were obtained and washed for each days titration work. A 1percent suspension of cells was used in the titrations and a 5 percentsuspension for spot testing allantoic fluid for hemagglutination in theinfectivity and sterility tests.

Vaccinated and young developing chickens were held in the same quarters.The unvaccinated birds served as controls for the vaccinated ones, sincethey were bled prior to vaccination themselves and their sera examinedfor antibody against Newcastle disease by both the hemagglutinationinhibition (alpha method) and the serum neutralization test inembryonated eggs.

A given pool of Newcastle disease virus infected allantoic fluid wasthawed, and titrations of both hemagglutinin and infectivity were made.A 1:10 dilution of the virus was then prepared in a large sterile vesselwith sterile 0.85 percent sodium chloride so as to give a final volumeof two to three liters. This material was then poured into the reservoirof the heat exchanger which had been presterilized by steam underpressure and cooled prior to use, and compressed air was applied toinitiate controlled flow of the fluid. The fluid was heated to thehighest temperature level to be tested in 0.033 second, and desiredtemperatures were regulated at different intervals. Samples heated tothese temperatures in 0.033 second were collected in 500 ml. flaskscontaining marbles prefrozen in the freezer. The marbles served as acooling mechanism. Samples taken were between 20 to 30 ml. in volume.There was an inherent holding time of approximately 0.022 second due tothe sample collecting technique.

Each heated sample, representing the attainment of a differenttemperature level in 0.033 second time, was tested for itshemagglutinability and its infectivity :Eor ten-day-old embryonatedchicken eggs as shown in Table I using one degree intervals to pinpointthe temperature of inactivation. The sample at the inactivationtemperature (or the sample collected at that temperature level which wasunable to infect embryonated eggs) was then tested for its sterility.Sterility checks consisted of three blind passages of allantoic fluidfrom embryonated eggs inoculated with the sample from the heat exchangergiving no infection as reflected by hemagglutination in tenday-oldembryonated chicken eggs from Newcastle disease virus susceptible birds.If all three egg passages still proved negative for the appearance ofvirus then Newcastle disease virus susceptible four-week-old chickenswere vaccinated with the just inactivated sample and tested for theirimmunological response. Birds were vaccinated with 0.5 or 1.0 ml. of agiven sample intramuscularly as indicated.

The first batch to be tested (batch I) had an EID titer of (0.1 ml.)(10- As can be seen from Table I, after passage through the heatexchanger hemagglutination was completely lost at 174 F. and at 179 F.the ability to infect embryonated eggs was also lacking. Sterilitychecks on the 179 F. sample all proved negative for virus byhemagglutinationtesting of allantoic fluid from Newcastle disease virusfree embryonated eggs inoculated with the sample.

' Four-week-old Newcastle disease virus susceptible chicks werevaccinated iutramuscularly with 0.5 ml. of the 179 F. sample every sevendays for three weeks. The vaccinated birds were bled one, two, ten, andnineteen Weeks after the final vaccination, and their sera was examinedfor its ability to inhibit hemagglutination and to neutralize virus bythe serum neutralization test. Table 11 gives the hemagglutinationinhibition and serum neutralization titers. It can be seen from thistable that birds Nos. 6, 7, 8, 9, and 10 demonstrated a consistentlystrong immune response by the serum neutralization test through thenineteenth week. The hemagglutination inhibition titers covering thenineteen-week period were rather erratic; however, birds Nos. 6 and 7remained consistently strong throughout while birds Nos. 8 and 10responded strongly through the two-week period but began to fall offfrom the tenth week to the nineteenth week. .Hemagglutination inhibitiontiters at the nineteen-week period were 1:640 for bird No. 9, 1:320 forbirds Nos. 6 and 7, 1:160 for bird No. 8, and 1:10 for bird No. 10.

A second batch (batch II) was prepared in a like manner as described forthe first batch. The EID of the second batch prior to heating was (0.1ml.) (IO- Temperature heated to ranged from 172 F. to 176 F. Table IIIshows that hemagglutination was lost at 174 F. and that the 175 F.sample was unable to infect embryonated eggs. Sterility tests on the 175F. sample were all negative for virus by hemagglutination.

An evaluation of the immunogenic potential of the just inactivated or175 F. sample was attempted. Three groups consisting of five Newcastledisease virus susceptible birds each were arranged so that each bird inthe first group received one inoculation intramuscularly of 1.0 ml. ofthe sample; each bird in the second group received two inoculationsspaced three days apart; and each bird in the third group received threeinoculations three days apart. The birds in the one and two-inoculationgroups were bled at two, three, six, and fifteen weeks aftervaccination; and the birds in the three-inoculation groups were bled attwo, three, six, and fifteen weeks aftenvaccination; and the birds inthe three-inoculation group were bled two, six, and fifteen weeks afterthe final vaccination. All sera were tested for their ability to inhibithemagglutination and all samples were tested for their ability toneutralize virus by the serum neutralization test. The results arepresented in Table IV. It can readily be seen that on the basis of therelatively short term postvaccination period being reported here, asubstantial immune response appeared with birds Nos. 13 and 14 two weeksafter one vaccination. Three birds (Nos. 19, 20, and 23) of thetwo-inoculation group, for the same time interval, yieldedhemagglutination inhibition titers of 1:2560 or greater, and all fivebirds (Nos. 19, 20, 21, 22, and 23) had serum neutralization titersranging from 1X10 to 1x10 per ml. at this period. At the three-weekperiod all birds in the oneand twoinoculation groups had generallyconsistent serum neutralization titers ranging from greater than 3X10 to1x10 per ml. At the six-week period, serum neutralization titers rangedfrom greater than 2 10 to less than 2X10 per ml. for all three groups.Hemagglutination inhibition titers at the three-week period for theoneand two-inoculation groups and the two-week period for thethree-inoculation group were quite consistent at greater than 1:640 withbird No. 27 of the three-inoculation group yielding a 1:80hemagglutination inhibition titer.

At fifteen weeks one bird in the one-inoculation group yielded ahemagglutination inhibition titer greater than 122560, while two otherbirds in this group had titers of 1:640 and one of 1:320; three birds inthe second group yielded hemagglutination inhibition titers of 1:640,while one was found to be greater than 1:2560; and of the threeremaining birds in group three, one yielded a hemagglutinationinhibition titer greater than 1:2560 and two of 1:320. Two birds ingroup three died during the latter part of the experimental period andunfortunately were destroyed before the cause of death could bedetermined.

Batch II vaccine had been stored in the freezer from the time ofpreparation to four and one-half months and its immunogenic potencyre-evaluated. Twenty Newcastle disease virus susceptible chickens, fourweeks old, were vaccinated with 0.5 ml. of batch II which had beenstoredin the freezer for four and one-half months. The birds were bledfour and one-half weeks after vaccination and their sera were tested fortheir ability to inhibit hemagglutination and to neutralize virus in theserum neutralization test. Hemagglutination inhibition and serumneutralization titers for this experiment are reported in Table V.Eleven of the twenty birds yielded hemagglutination inhibition titersgreater than 1:256'0, one at 1: 1280, one at 1:320, and two at 1: 160.The remaining five birds had hemagglutination inhibition titers lessthan 1:160. Thus, 75 percent of the birds in this experiment yieldedsignificant responses based on the hemagglutination inhibition testsabove. Serum neutralization titers ranged from greater than 3X10 to 1X10for thirteen of the vaccin-ated birds, while the remaining seven hadtiters of less than 3x10 one of which was 5x10 and three of 1X10 to 4x10It is important to emphasize that the dosage was 0.5 ml. per bird andthat only one vaccination was used, and also that the vaccinepreparation had been stored for four and one-half months at freezertemperatures.

Table I.-Inactivati0n of Hemagglutination and Infectivity by Heating toVarious Temperatures in 0.033 Second BATCH I Titrations ofhemagglutination Infectivity for Temperature (dilution, of sample)embryonated eggs virus heated (egg N0.)

second to Unheated 0 0 F 0 0 0 0 a 0 0 O 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 O 0 0 0 0 0 0 0 0 0 0 0 0 0 O 0 0 0 0 0 0 U 0 0 eachof the specified sample.

Table II.Hemagglutination Inhibition and Serum Neutralization Resultsfor Sera From Newcastle Disease Virus Susceptible Chickens VaccinatedWith the 179 F.

Inactivated Sample of Batch I Weeks after final vaccination Bird N0. 1week 2 weeks weeks 19 weeks HI SN/ml. HI SN/ml. HI SN/mll HI SN/ml.

6 111280 6X10 1 :20,480 3X10 1 :320 7x10 1:320 5X10 7 1:1280 2X1010:20,480 3Xl0 1z320 1X10 1:320 5X10 8 1 :1280 1 X10 1 :20,480 3 10 1:320 4X10 1:160 5X10 9 1 :80 1 X10 1 :80 8X10 1:20 4X10 1:640 5X10 101z1280 1X10 1:20,480 4X10 1:40 2X10 1:10 5X10 HI=HeInagglutinationinhibition.

Norn-Pooled sera from five control birds was run coincidently with thetest and no inhibition of hemagglutination or neutralization of testvirus was evident.

Table lII.Inactivati0n of Hemagglutination and Infectivity by Heating toVarious Temperatures in 0.035

Second SN Serum neutralization.

Table V.Hemagglutinati0n Inhibition and Serum Neutralization Results forSera From Newcastle Disease Virus Susceptible Chickens Vaccinated With0.5 ml. of the 175 F. Sample of Batch 11 After Storage at Freezer BATCHII Temperature for Four and One-half Months T W Tltlalglrlls ofhemagglultination Infgetivitx fgr empera e virus ution o samp e em ryonae heated in 0.033 e gib e i v ib second to- Bird No.

1:2 1-4 1 8 1:16 1 32 1=e4 1:128 1 2 3 4 HI titer SN titer Unheated 0135 F. (Preh t)- 0 3? 118 52%;: F 0 0 30 1 25 0 2 100 0 0 40 21 10 51Q20 0 0 0 0 0 0 0 0 41 12560 6X10 0 0 0 0 0 42 1140 6x10 43 1225G0 3X10:+=hemagglutination. 0=absence of hemagglutination. it Norm-All eggs forinfectivity studies were inoculated with 0.1 ml. 46 122560 3 10 47 1Z104X10 each of the specified sample. 48 1z2560 1x107 Table IV.Hemagglutinati0n Inhibition and Serum Neutralization Results for SeraFrom Newcastle Disease Virus Susceptible Chickens Vaccinated With the175 F. Inactivated Sample of Batch lI ONE INOCULATION Time after finalvaccination Bird No. 2 weeks 3 weeks 6 weeks 15 weeks HI SN/ml. HISN/ml. HI SN/ml, HI SN/Iil].

1 :2560 3X10 1 :640 1X10 1 :320 1x10 1:640 5 10 112560 5X10 1 :640 1X101:40 2 X10 1:10 5X1O 1 3X10 1 :640 5x10 1 :320 2 l0 1:640 5 10 1:40 1 101:640 3X10 l:320 5x10 1:320 8X10 1 :20 6x10 1z640 3X10 1 :320 2X101z2560 5 10 TWO INOCULATIONS 1X10 1z640 3X10 1z320 1X10 122560 5X10 1X1012640 3X10 1:320 2Xl0 1:640 5 10 1x10 12640 3X10 1z320 6x10 1:160 2X103X10 1:640 4X10 1:320 1x10 1:640 5X10 3X10 12640 3X10 l:320 2 10 1:6407x10 THREE INOCULATIONS 1=e40 3 10 i=320 2 10 1:640 3X10 1Z320 1X10 1:640 3 10 1 :320 1 X10 1: 4X10 1:160 1X10 1:640 3Xl0 1:320 1X10 1 Birdsdied during the fifteen-week period. HI=Hen1agglutination inhibition.SN=Serun1 neutralization.

N0'1E:P0Oled sera from five control birds W2 .S run coincidently withthe test and no inhibition of hemagglutination or neutralization of testvirus was evident.

8 Table V-Contmued V v The particular temperature capable ofinactivating the live virus microorganisms will depend mainly upon the nan h g particular virus being inactivated and the time during Bird NOafter Vaccination which the virus suspension is heated. Using apreselected time such as the 0.033 second selected in the HI titer SNtiter above examples, the temperature at which the virus becomesinactivated or just inactivated can readily be deg3 termined by thoseskilled in the art in the same manner 51 lil60 5x10 in which theinactivating temperatures were determined i38 g in the above examples. Apreliminary run from a me 54- 1=25s0 a 10 heat temperature of about 135F. at 5 degree intervals 22 g3 might be helpful before pinpointing theexact inactivating temperature by heating the virus suspension at 1degree HI=Hemagglutination inhibition. SN: Serum neutralizamtel'vals-All temperatures below the inactivating temtion. perature theinfectivlty properties of the virus suspensions ssiratirsairraaassessments;are are still retained- Heating e virus to eneutralizationof test Virus was evident peratures beyond the ust inactivatingtemperatures is EXAMPLE 2 not necessary or even desirable since the useof higher temperatures will generally tend to destroy the antigenic TheVll'llS of influenza A-strain PR8 was sub ected to the structure f h iidentical high temperature-short time thermal inactivation Th i i hi h hvirus suspensions are h t d t treatment as set forth above with respectto the Newcastle th ina tivating temperature should be as rapid as pdisease virus using a 1:10 dilution of chick emb sible but sufficientlyslow so as to eiTect the desired inlantolc fluld Suspenslon of theVlrusactivation. Generally, it is advisable to heat to the inac- TableVI Shows the inactivation of hemagglutinafioll tivating temperaturewithin a fraction of a second. and infectivity of the influenza virussuspension. The re- Aft the virus vaccines h b h d to h i 811118 w hainfectivity of the Virus yactivating temperature they are cooled bysuitable means chick embryos was lost at such as by collecting theheated virus suspension in flasks A group of fi four'week-olfl Chickswere glvel'l three containing marbles stored in a freezer until justprior to 1 ml. vaccinat1ons of this inactivated sample spaced onecollection f the suspension Generally, the mm heatweek apart. These fivebirds along with unvaccinated j d mi i i 1 h two i control birds werethen bled four days, ten days, and fourteen week following the finalVaccination Their Sera Tabla VI.-Inacnvat10n of Hemagglutmatlon andInfec- Was examined for its ability to inhibit hemagglutination tlwty oflnflueriza A'Stmm PR8 By Heatmg to Various and to neutralize virus inthe serum neutralization test. Temperatures m Second The results ofthese tests are shown in Table VII. Four days after the finalvaccination or twenty-five days after g g g fg Qfhemaggluflnation ct ivity for em e r the first vaccination, all five chickens yielded HItiters 3 65 $6.316 lumen otsample) mate eggs (egg ranging from 1:640 toH160 and SN titers ranging from 142 1 4 18 n6 1 2 3 4 5X10 to 1x10 Tendays after the final vaccination 40 the HI titers had not changedappreciably, but the SN titers dropped slightly and ranged from 3 X10 to1x10 A-t fourteen Weeks the SN titers ranged from 7X 10 to 3 X10 The H1titers were still quite consistent. Chicken No. 31 died during thelatter part of the experiment and unfortunately was destroyed before thecause of death could be ascertained.

The results of this experiment show that the inactivated influenzaSuspension was capable of Stimulating an +=hemagglutinsti0n. 0=absenceofhemagglutination. mune respon e in the a cinated bird The response ofNOTE.A11 eggs I01 infeetivity studies were inoculated with 0.1 ml. thechickens to the vaccine held up for the fourteen-week each of thespeclfied samp Unheated" 160 F ooo++++++ oooool'bH-H-docaocccai'i"ooocaoooo ooocvcocoo cccocac+++ oooooc oooooooo'i COQOQOOO+ period eventhough there was a rather significant drop Table Vll.Hemagglutinati0nInhibition and Serum in H1 titer after the ten-day test period.Neutralization Results for Sen: From Chickens Vac- The inactivated virusof influ nza. A- fl' ill PR Was cinated With 162 F. Inactivated Sampleof Influenza found to p ess e li y o produce a significant A-Strain PR8(3 Shots of 1.0 MI. Each One Week mune response in four-week-old chicks.Even though Apart) the SN and HI titers dropped during the fourteen-weekholding period, SN titers in the range of 10 were detected as well assignificant HI titers. Tune after final vaccmgmn The virus suspensionstreated in the above examples 50 were heated to the inactivatingtemperatures almost in- Bird ways 10 days 14 weeks stantaneously (0.033second). The time limit of 0.033 second was chosen because ofconvenience in view of the HI SN/ml' HI SN/ml HI SN/ml' equipment beingused to heat the virus suspensions. The virus suspensions were alsopreheated to a temperature of 135 F. by means of heat exchange from aportion of the heating tube before being heated to the inactivatingtemperature. The virus suspensions were heated to the inactivatingtemperature y heat exchange from a HI=Hemagglutination inhibition.SN=Serum neutralization. foot Section of the heating tube at thedischarge end of NorE.Poo1ed sera from five control birds was runcoincidently the heating apparatus which was designed so that the withthe test and no inhibition of hemagglutination or neutralizationtemperature could be controlled as desired by electrical test was means.The apparatus was calibrated to heat to the de- We claim:

sired inactivating temperature in 0.033 second and to 1. The method oftreating a suspension of virus microyield 1100 ml. of inactivated virussuspension per minute. organisms to render the microorganismnon-infective 1:160 1X10 1:160 1X10 12320 7X10 1:640 4X10 1t60 1x101:160 3X10 1:320 5X10 1:320 3X10 1: 5X10 l: 160 1x10 12320 7X10 1:1601X10 1: 320 3x10 11320 7x10 9 10 without significantly impairing theantigenic properties 2. The method of claim 1 in which the suspension ofthereof which comprises: virus microorganisms is heated at thetemperature at which the microorganism does not exhibit infectivity butretains substantially maximum antigenicity in about .033 5 second.

3. The method of claim 1 in which the virus suspension of microorganismsis Newcastle disease virus.

4. The method of claim 1 in which the suspension of virus microorganismsis influenze.

(1) heating a sample of a suspension of virus microorganisms by aheat-exchanger at various different relatively high temperatures inabout a fraction of a second;

(2) testing the microorganisms after heating at each temperature todetermine the infectivity thereof;

(3) continuing the heating of samples of suspensions of virusmicroorganisms as described in step 1 so long 10 R C't th t as thesample stall exhibits infectivity at the temperadefences 1 ed m e fileof thls paten tures employed until a temperature is obtained at UNITEDSTATES PATENTS which the microorganisms do not exhibit infectivity2,202,435 Shoetensack May 28, 1940 when tested and which microorganismsretain sub- 2,324,646 Rake July 20, 1943 stantially maximumantigenicity; and 15 FOREIGN PATENTS (4) then heating an entire batch ofa suspension of virus microorganisms propagated under substantially the160542 Australia 1955 same conditions as the microorganism contained inOTHER REFERENCES the test samples in about a fraction of a second at 20JAMA, 158, May 14, 1955, p. 138,

the temperature determined according to Step 3 at Zinssers Textbook ofBacteriology, Appleton-Centurywhich the microorganism does not exhibitinfectivity Crafts, N.Y., 9th ed., 1948, pp. 14, 15, 698, 699. butretains substantially maximum antigenicity. PSEBM, 93 -1, October 1956,pp. 51-52.

1. THE METHOD OF TREATING A SUSPENSION OF VIRUS MICROORGANISMS TORENDEER THE MICROORGANISM NON-INFECTIVE WITHOUT SIGNIFICANTLY IMPAIRINGTHE ANTIGENIC PROPERTIES THEREOF WHICH COMPRISES;